This invention relates to apparatus for cooling a biological or medical specimen and, in particular, to apparatus for cryogentic-fixation of a specimen for microscope inspection. Cryogenic-fixation of a specimen involves the injection of the specimen at a rapid velocity, suitably between 5 and 15 m/sec, into a cooling liquid which may, for example, be pre-cooled to -190.degree. C.
Cooling baths containing liquids at temperatures between -100.degree. C. and -190.degree. C., and with volumes between 5 and 100 ml, are used for numerous specimen-preparation operations, particularly for the process of shock-freezing or "cryogenic-fixation" of small biological or medical specimen for subsequent microscopic examination. Suitable preparation of the specimen which has been introduced into the cooling liquid can be achieved only when as much heat as possible is abstracted from the surface of the specimen within the shortest possible time. This applies particularly for biological or medical specimens which have not been pre-treated, that is, specimens which have not been subjected to a preliminary fixation and/or freezing-protection treatment. For such specimens, the cooling rate alone determines whether artificial separation of the water-rich plasmatic phases takes place (separation would render meaningful microscopical examination impossible) or whether the specimen freezes to a true-to-life, glassy form ("vitrification" takes place at cooling rates greater than 10,000.degree. C./sec).
The cooling rates which are required for vitrification are obtained only in an edge zone of the specimen, this zone having a depth of 30 um at the most. The depth of the perfectly vitrified edge zone is determined essentially by the temperature and the specific properties of the cooling medium, and by the velocity at which the specimen enters the cooling medium. Liquified propane which has been cooled to a temperature only slightly above its freezing point (-190.degree. C.) offers the best known conditions for vitrification. Using this cooling medium, initial cooling rates of the order of 100,000.degree. C./sec are obtained, but only if the specimen is injected into the liquid at velocities exceeding 5 m/sec.
The liquefaction, cooling, and storage of propane, under constant-temperature conditions, presents few problems. However, the injection of the specimen presents considerable problems and hazards. Since the specimen is injected in at a comparatively high velocity, up to 15 m/sec, a considerable quantity of the cooling liquid may splash out of the cooling bath. Typically, the total quantity of cryogen is kept below 100 ml in order to minimise the hazards associated with propane, i.e., fire, and the danger of explosion. The diameter of the specimen, and/or of the injector, typically exceeds 3 mm, and it is usually necessary that the specimen should travel a distance of 5 to 10 cm in order to achieve adequate cooling. While it is possible to use a non-flammable cooling media, such as halogenated hydrocarbons, (e.g. FREON 13, which has a boiling point of -185.degree. C.), these provide a 20% lower cooling rate and other problems still remain. The splashing of the cooling liquid will cause severe burns if the liquid strikes the skin.
More than 90% of the cooling medium is often lost from the cooling bath when specimens are introduced at velocities greater than 10 m/sec, the specimens and specimen holders are of conventional diameter (approximately 3 to 5 mm), and the injectors are of conventional diameter (approximately 4 mm). Insufficient liquid usually remains in the cooling both to cool further specimens, even when the specimens are small, and the heat capacities of the specimen-holder and injection device are low. When a specimen and specimen holder is insufficiently cooled, the quantity of heat which remains in the interior of the specimen, and/or in the specimen holder/injector assembly, causes secondary heating of the surface of the specimen following the initial superficial cooling. Hence, an artifical separation of the initially perfectly vitrified surface occurs. In order to be able to enhance the quality of the cryofixation by means of greater injection velocities, as well as for safe operations, it is desirable to adopt precautions to prevent splashing of the cooling medium even when the injection velocities are comparatively high.